CN107828689B - A strain of Bacillus amyloliquefaciens S170 for controlling rice blast - Google Patents

Abstract

The invention discloses a Bacillus amyloliquefaciens for controlling rice blast and application thereof. The bacillus amyloliquefaciens of the invention has broad-spectrum and high-efficiency control effects on rice blast, and can be used for preventing and controlling rice blast of various strains, especially leaf blast and panicle blast.

Description

A strain of Bacillus amyloliquefaciens S170 for controlling rice blast

technical field

The invention relates to the field of microorganisms, in particular to a strain of Bacillus amyloliquefaciens for controlling rice blast and its application.

Background technique

Rice is an important food crop in the world. Its output accounts for more than 1/4 of the world's total food production, more than half of China's total food production, and is the staple food that more than half of the world's population depends on. Rice blast (Rice Blast) is a kind of sudden and strong worldwide fungal disease caused by Ascomycete Magnaporthe oryzae (T.T.Hebert) Yaegashi&Udagawa (asexual state: Pyricularia oryzae). The disease can cause harm to rice all year round, and its harm spreads all over the various parts of rice, including seedling rice blast, leaf blast, leaf pillow blast, node rice blast, ear neck blast, branch blast and grain blast. Rice blast is widely distributed and occurs in 85 countries around the world. It is also one of the most serious rice diseases in the northern and southern rice regions of my country. Rice blast causes 10-30% rice yield loss every year in the world, and the annual yield loss can feed 60 million people in the world.

At present, the control of the blast fungus mainly adopts the selection of disease-resistant varieties and chemical control. Long-term use of chemical agents not only increases the pesticide residues in agricultural products, but also causes serious pollution of the ecological environment, destroys the balance of the ecosystem, and threatens food safety. Bacillus (Bacillus) can not only effectively control plant diseases, but also has the advantages of being safe for humans and animals, not easy to produce resistance to plant pathogens, strong stress resistance, and promoting plant growth. It is an important biocontrol bacterium in the control of rice blast. Bacillus colonizes rice plants, produces antibacterial active substances to inhibit the growth of blast fungus, induces rice to develop disease resistance, has a growth-promoting effect on rice plants, and can restore rice yield loss. Bacillus can be used to prepare biocontrol agents for the prevention and treatment of rice blast in the southern and northern rice regions of my country, and has guiding significance for the biological control of rice blast in the sustainable development of the rice industry. Bacillus can prevent and control the sudden damage of rice blast in the period when chemical pesticides cannot be applied, minimize the yield loss caused by rice blast, and will not increase the residue of toxic substances in rice, let alone pollute the environment.

The spores of Magnaporthe grisea are the main infection organs. After the spores mature, they produce germ tubes, which attach to the surface, and the germ tubes directly enter from the rice epidermis through natural orifices or infection plugs. Among them, the formation of appressoria is one of the key steps for successful invasion. It must generate enough pressure to mechanically penetrate the host keratin layer to complete the invasion process, and melanin is the prerequisite for generating appressorium pressure. Melanin is deposited in a very small area on the side, and finally the attachment cells are formed to complete the invasion process. By successfully colonizing the plant rhizosphere, body surface or body, Bacillus competes with Magnaporthe grisea for nutrients around the plant, secretes antibacterial substances to inhibit the growth of pathogenic mycelia, germination of conidia, deforms appressoria and dilutes melanin, and at the same time Induce the rice defense system to resist the invasion of blast fungus, so as to achieve the purpose of biocontrol. Bacillus can produce chitinase, sugar decomposing enzyme, cell wall degrading enzyme, etc. to promote the dissolution of the mycelial cell wall of Magnaporthe grisea, especially those mycelia that are old or even close to death are more likely to dissolve. Idris et al. (2007) reported that the fermented broth of Bacillus amyloliquefaciens FZB42 and FZB45 had obvious growth-promoting effect on rice because the fermented broth contained the plant hormone IAA, which proved that IAA is the main substance that FZB42 promotes plant growth. Ng et al. (2012) mixed Bacillus amyloliquefaciens UPMS3 and a variety of microbial fertilizers, which significantly improved the biocontrol effect on panicle blast and leaf blast after being used in the field, and could reduce rice yield loss by 31%. The growth-promoting effects of tree number, leaf area and plant height were significantly improved. Li Shuangdong et al. (2015) reported that the fermentation broth of Bacillus amyloliquefaciens RL263 had a maximum control effect of more than 70% on rice leaf blast, which could significantly increase rice yield. Huang Yuanhui et al. (2016) isolated Bacillus amyloliquefaciens BCHN-15 from the coastal waters of Dalian by using the confrontation method and the Oxford cup method. After 48 hours of treatment with Magnaporthe grisea, the dry mycelium weight of Magnaporthe grisea decreased by 74.36% compared with the control group. %. The antibacterial components produced by the fungus can destroy the cell membrane of the rice blast fungus, causing deformity and rupture of the hyphae. The protease and cellulase produced by Bacillus amyloliquefaciens WH1G screened by Gu Chunyan et al. (2016) had an inhibitory effect of 91.79% on Magnaporthe grisea, and the sterile filtrate could reduce the germination rate of Magnaporthe grisea spores, with abnormal mycelium distortion and obvious branching Reduced and produced a large number of vesicles. Wang Fafa et al. (2017) sprayed Bacillus amyloliquefaciens T429, JT84, Lx-11, and B-916 dry suspension concentrate 75g/667m ² at the rice breach stage, and the control effects on rice panicle blast were 85.2%, 79.3%, respectively. %, 79.6%, 78.9%; using 4 dry suspensions of Bacillus amyloliquefaciens to soak rice seeds, when diluted 500 times, it is safe for rice seeds and has different degrees of growth-promoting effects.

Utilizing enzyme engineering and protein engineering technologies, the research and application of new Bacillus biocontrol agents with protein as the active ingredient in the near-harvest period of crops can maximize the recovery of the rice industry’s yield caused by sudden diseases in the near-harvest period loss. The research and application of Bacillus to control rice blast is the need for sustainable development of rice production, and can meet the requirements of the United Nations World Food Organization for the development of food security. Bacillus strain with high production of active substance.

Contents of the invention

The object of the present invention is to provide a Bacillus amyloliquefaciens strain that has good control effects on rice blasts of different varieties of rice, especially leaf blasts and panicle blasts, and has growth-promoting and production-promoting effects.

For reaching this purpose, the present invention adopts following technical scheme:

In the first aspect, the present invention provides a kind of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) S170, which is preserved in the General Microorganism Center (CGMCC) of China Microorganism Culture Collection Management Committee, and the preservation date is May 22, 2017, and the preservation number is CGMCC No. 14179.

In the second aspect, the present invention provides a bacterial agent, which comprises the Bacillus amyloliquefaciens S170 as described in the first aspect or the sterile supernatant obtained by culturing the Bacillus amyloliquefaciens S170. .

In a third aspect, the present invention provides a method for controlling rice blast, which comprises applying the Bacillus amyloliquefaciens S170 as described in the first aspect or the bacterial agent as described in the second aspect to rice crops.

In the method for controlling rice blast of the present invention, the bacterial agent is the culture solution of the Bacillus amyloliquefaciens S170.

In the method for controlling rice blast of the present invention, the rice blast is leaf blast and/or ear blast.

In the fourth aspect, the present invention provides the use of Bacillus amyloliquefaciens S170 as described in the first aspect or the bacterial agent as described in the second aspect in the control of rice blast.

In the use of controlling rice blast in the present invention, the rice blast is leaf blast and/or neck blast.

The beneficial technical effect of the present invention is:

1. Compared with the existing Bacillus amyloliquefaciens for controlling rice blast, the Bacillus amyloliquefaciens of the present invention has a higher control efficiency, which is reflected in that similar or better control effects can be obtained with less spores.

2. Compared with the existing Bacillus amyloliquefaciens for preventing and treating rice blast, the Bacillus amyloliquefaciens of the present invention has a wider spectrum of control effect.

3. Compared with the existing bacillus amyloliquefaciens for preventing and treating rice blast, the bacillus amyloliquefaciens of the present invention has a better control effect on rice leaf blast and panicle blast.

4. Compared with the existing Bacillus amyloliquefaciens for preventing and treating rice blast, the Bacillus amyloliquefaciens of the present invention has a better effect on promoting growth and production of rice.

Description of drawings

Fig. 1 is the culture form of Bacillus amyloliquefaciens S170 strain on the NA medium plate.

Fig. 2 is a plate confrontation picture of Bacillus amyloliquefaciens S170 strain and Magnaporthe grisea P131.

Fig. 3 is a plate confrontation picture of Bacillus amyloliquefaciens S170 strain fermentation medium, sterile supernatant and Magnaporthe grisea P131.

Figure 4 is an electron microscope picture of the inhibition of the Bacillus amyloliquefaciens S170 strain on the P131 infection structure of the rice blast fungus. Subsequent electron microscope image.

Figure 5 is an electron microscope picture of the inhibition of the bacterial supernatant of Bacillus amyloliquefaciens S170 on the infection structure of Magnaporthe grisea. Electron micrographs of conidia and hyphae after the infection structure of Magnaporthe grisea P131 was inhibited.

Fig. 6 is a picture of detection of biocontrol active substances of Bacillus amyloliquefaciens S170 strain.

Figure 7 is the inhibition result of the volatile substances produced by Bacillus amyloliquefaciens S170 on the rice blast fungus P131. Inhibition results of P131.

Detailed ways

The technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and through specific implementation methods.

Embodiment 1: Screening and fermentation culture of Bacillus amyloliquefaciens S170 bacterial strain

(1) Sample collection: The sample was collected from the rhizosphere soil of rice variety Ningjing 48 at Shaqu Village, Yaofu Town, Pingluo County, Ningxia Hui Autonomous Region. Pull out together with the root system of rice, pack it in a polyethylene plastic bag, and bring it back to the laboratory for isolation.

(2) Isolation of Bacillus amyloliquefaciens: Weigh 1 g of rhizosphere soil, put it into 100 mL of sterile water, shake and mix at room temperature for 1 hour, dilute with sterile water according to 10, 10 ² and 10 ³ gradients, and use a micropipette Take 200mL/dish of the gradient dilution solution and spread it on the NA solid medium plate with a spreader stick. After incubating for 48 hours in a 37°C incubator, pick a single colony with the inoculation needle and streak it on the NA solid medium plate with a sterile toothpick to obtain purified The final bacterial single colonies were stored in 40% glycerol and stored in a -80°C ultra-low temperature refrigerator for later use.

(3) Screening of Bacillus amyloliquefaciens: select healthy rice leaves, cut 5 cm long leaf segments, put them into the Bacillus S170 fermentation broth, shake and colonize for 1 hour, and obtain the leaves after colonization; lay filter paper on the bottom of the culture dish, and then Place the leaves after colonization on moist filter paper, and after colonization and culture for 24 hours, use a sterilized toothpick to lightly puncture the surface of the leaf segment without injuring the cuticle of the leaf segment. Draw the spore suspension of Magnaporthe grisea and inoculate them on the 5 points of the puncture wound respectively, and obtain the leaves after inoculation. After cultivating in an artificial climate box for 24h, 48h and 72h, investigate the number and area of rice blast lesions at the inoculation point, and select the inoculation point without disease Bacillus bacillus with light color and small area of spots or lesions should be stored at low temperature for later use.

Broth medium (NA): peptone 10.0g, beef powder 3.0g, sodium chloride 5.0g, agar 15.0g, pH 7.3±0.1, sterilized at 121°C for 20min.

LB liquid medium: 5g of yeast extract, 10g of peptone, 5g of sodium chloride, 1000mL of water, pH7.4-7.6, sterilized at 121°C for 30min. LB solid medium: add 15g agar powder on the basis of liquid medium.

Oat medium: 30g of oat flakes, 17-20g of agar powder, add 1000mL of water, heat in a boiling water bath for 1h, filter through gauze, add water to make up 1000mL, add agar powder, subpackage and sterilize (121°C, 20min).

After identification, the characteristics of the Bacillus amyloliquefaciens strain are as follows: white, translucent, smooth and moist colonies, slightly raised, round and neat colonies. The cells are Gram-positive bacteria, rod-shaped, (0.7-0.9) μm × (1.8-3.0) μm, growing in chains, and relying on perinatal flagella for movement. The spores are round (0.6～0.8) μm×(1.0～1.4) μm, middle or terminal, and the cyst is not enlarged.

The inventor has deposited this strain in the General Microorganism Center (CGMCC) of the China Committee for the Collection of Microbial Strains, with the preservation date being May 22, 2017, and the preservation number being CGMCC No.14179.

Embodiment 2: the biological activity assay of Bacillus amyloliquefaciens S170 bacterial strain:

(1) Antibacterial determination of Bacillus amyloliquefaciens S170 strain on Pyricularia grisea: Place Pyricularia grisea P131 bacterial cake in the center on the tomato oat plate medium, and inoculate a single colony of Bacillus at a distance of 2 cm on both sides of the bacterial cake after growing for 4 days Bacteria cakes were used as confrontation culture, and sterile water was used as control, and cultured in the dark at 28°C in an incubator. Each treatment was repeated 3 times. After 2 days, the diameter of the pathogenic bacteria colony and the zone of inhibition were measured, and the results are shown in Table 1.

(2) Bacillus amyloliquefaciens S170 aseptic supernatant antibacterial determination of rice blast fungus: Place blast fungus P131 fungus cake in the center on the tomato oat plate medium, place sterile Oxford cups at a distance of 2 cm from both sides of the fungus cake , the fermentation broth and sterile supernatant of Bacillus were added to the cup for confrontation culture, and sterile water and sterile LB liquid medium were used as controls, respectively, and placed in an incubator at 28°C for dark culture. Each treatment was repeated 3 times. After 5 days, the diameter of the pathogenic bacteria colony and the zone of inhibition were measured, and the results are shown in Table 1.

Preparation of the sterile supernatant of the strain: Inoculate the single colony of the test strain S170 that has been activated for 24 hours into LB liquid medium, shake culture at 37°C and 200rpm/min for 48h, and ferment the culture medium at 4°C and 10000rpm/min Centrifuge for 20 minutes to remove bacteria, and the supernatant is sterilized by filtration (0.22 μm) to obtain a sterile supernatant.

Table 1 The confrontation culture of Bacillus amyloliquefaciens S170 strain and rice blast fungus

Note: The experimental data in the table are the average value of three repetitions.

(3) Determination of the control effect of isolated leaves of Bacillus amyloliquefaciens S170: select healthy rice leaves, cut off 5 cm long leaf segments, put them into the Bacillus S170 fermentation broth, 200rpm/min, shake and colonize at 28°C for 1h, and obtain colonization Back leaves: Lay filter paper on the bottom of the petri dish, place 5 sterilized toothpicks on the moistened filter paper, place the leaves after colonization on the toothpicks, after colonization and cultivation for 20 hours, lightly stab the surface of the leaf section with a new sterilized toothpick, Without injuring the cuticle of the leaf segment, puncture 5 points on each leaf segment, and then use a pipette gun to inoculate the spore suspension of Magnaporthe grisea on the 5 punctured points respectively to obtain the inoculated leaves, and place them in an artificial climate box. After 24h, 48h and 72h of culture, the number and area of rice blast lesions at the inoculation point were investigated, and the control effect was calculated. The inoculated petri dish was cultured in an artificial climate chamber, the parameter settings were 28° C., RH (humidity) 70%, 4 hours of light per day, and 4400 Lux of light intensity. The results are shown in Table 3.

Grading standard for indoor investigation of rice blast leaf blast: according to the grading standard for the evaluation of rice blast resistance by the International Rice Institute (Table 2)

Table 2 is the grading standard of indoor investigation of rice blast leaf blast

Calculation formula of biological control effect:

Table 3 Determination of the control effect of Bacillus amyloliquefaciens S170 on detached leaves of Magnaporthe grisea

Note: The least range method (LSD) was used for significance analysis (P≤0.05±standard error).

(4) Biocontrol effect of Bacillus amyloliquefaciens S170 strain under potted conditions in greenhouse: select healthy rice variety D10, disinfect with 1% sodium hypochlorite solution for 10 minutes, rinse with 75% alcohol for 3 times, rinse with sterile water for 5 times, and artificially climate at 28°C Box germination, when the bud grows to 1cm, it is sown in a small plastic bucket (18cm in diameter at the bottom of the bucket * 21cm in height * 24.5cm in diameter at the top of the bucket). After 28 days of planting, the fermentation liquid of the bacillus to be tested was sprayed, the concentration of the spores was 1×10 ⁸ CFU/mL, 20 mL was sprayed in each bucket, and each treatment was replicated 4 times. After 24 hours, the spore suspension of Magnaporthe grisea was inoculated with a spore concentration of 1×10 ⁶ CFU/mL. After 7 days, the occurrence of leaf blast was investigated, and the disease index and control effect were calculated.

Table 4 Determination of the control effect of Bacillus amyloliquefaciens S170 on leaf blast under greenhouse conditions

deal with Disease index Control effect (%) S170 3.13±1.05bB 80.57±0.45aA Tricyclazole 2.98±0.14bB 81.51±0.69aA Clear water control 16.09±0.43aA -

Note: The least range method (LSD) was used for significance analysis (P≤0.05±standard error).

(5) Field control effect test of Bacillus amyloliquefaciens S170 strain

^The field efficacy test of Bacillus S170 was carried out at the rice planting base of Ningxia Heyin Modern Agriculture Co., Ltd. in Yaofu Town, Pingluo County, Ningxia Hui Autonomous Region. ml, the average area of each test plot is 50m ² , and 4 replicate plots were used to investigate the control effects of Bacillus S170 on leaf blast and panicle blast respectively. According to the "Guidelines for Field Efficacy Tests of Pesticides", the grading standards for rice blast leaf blast investigation are: level 0: no disease; level 1: brown spots the size of a needle tip; level 3: small, round and slightly elongated brown necrosis Gray spots, 1-2mm in diameter; Grade 5: typical rice blast spots, with damage area less than 10%; Grade 7: typical rice blast spots, damage area is 26%-50%; Grade 9: full leaf death. See Table 5 for the investigation methods of panicle blast, and Table 6 and Table 7 for the results.

Table 5 is the grading standard of rice blast panicle blast investigation

series survey criteria 0 No neck blast, grain blast and branch blast 1 There are grain blast and branch blast, the number of shriveled kernels is less than 5% 2 There are grain blast and branch blast, the number of shriveled kernels is 5-10% 3 There are grain blast and branch blast, the number of shriveled kernels is 10-20% 4 There are grain blast and branch blast, the number of shriveled kernels is 20-30% 5 There are panicle blast, grain blast and branch blast, and the number of shriveled kernels is 20-30% 6 Shrunken kernels caused by panicle blast > 80%

Calculation formula of biological control effect:

Table 6 Determination of field control effect of Bacillus amyloliquefaciens S170 strain on leaf blast

deal with Disease index Control effect (%) S170 8.52±0.53bB 73.19±0.96aAB green health 7.75±0.29bB 75.62±0.65aA Tricyclazole 8.44±0.19bB 73.45±2.01aA Clear water control CK 31.79±0.25aA /

Note: The least range method (LSD) was used for significance analysis (P≤0.05±standard error).

Table 7 Determination of field control effect of Bacillus amyloliquefaciens S170 strain on panicle blast

deal with Disease index Control effect (%) S170 11.79±0.66cB 74.82±0.41aA green health 12.25±0.73cB 76.25±0.37aA Tricyclazole 12.06±0.51bcB 74.18±1.08aAB Clear water control CK 46.86±3.06aA /

Note: The least range method (LSD) was used for significance analysis (P≤0.05±standard error).

(6) Growth-promoting ability of Bacillus amyloliquefaciens S170 strain on rice seedlings: select healthy rice seeds Ningjing No. 43 with consistent growth and soak them in the fermentation medium of Bacillus S170 for 1 hour, then take them out and place them in a culture medium covered with filter paper. Cultivate in an artificial climate box at 28°C and RH 70% in a dish, and investigate the number of white seeds after 48 hours. Each treatment was replicated 3 dishes. Take healthy and consistent rice seeds and soak them in the fermentation broth of Bacillus S170 for 1 hour, then sow them in rice seedling ponds, and investigate the root length and plant height of rice seedlings after growing for one month. The results are shown in Table 8.

Table 8 Growth-promoting ability of Bacillus amyloliquefaciens S170 to rice seedlings

Note: The least range method (LSD) was used for significance analysis (P≤0.05±standard error).

(7) The production-promoting ability of Bacillus amyloliquefaciens S170 strains on rice plants: Take healthy and consistent rice seeds Ningjing No. 43 and soak them in the fermentation broth of Bacillus S170 for 1 hour, and then sow them in rice seedling ponds. Spray the fermentation culture solution of Bacillus S170 at the transplanting stage, tillering stage, full-heading stage, and yellow maturity stage. When harvesting, the rice plants are pulled up together with the root system, and the effective panicle number, panicle length, grain number, and total weight of the rice plants are investigated. , Empty grain rate and 1000-grain weight, the results are shown in Table 9. The influence of Bacillus amyloliquefaciens S170 strain on rice quality was detected, and the results are shown in Table 10.

Table 9 The growth-promoting ability of Bacillus amyloliquefaciens S170 to rice plants

deal with Effective spike number/hole Spike length (cm) Empty ratio (%) Thousand grain weight (g) S170 11±2.03aA 207.13±9.05abAB 4.76±0.33bB 28.41±0.93aA green health 12±0.70aA 224.17±9.95aAB 5.87±0.29bAB 27.82±0.46aA Tricyclazole 10±0.72aA 186.14±1.18bcA 5.43±1.27bAB 29.45±0.03aA clear water 9±0.72aA 180.47±0.57cB 7.39±0.95aA 27.96±0.31aA

Note: The least range method (LSD) was used for significance analysis (P≤0.05±standard error).

Table 10 Effect of Bacillus amyloliquefaciens S170 on rice quality

deal with Brown rice rate Milling rate Finished rice rate Chalky Grain Rate Chalkiness S170 87.77±0.24abA 80.97±0.27aA 69.50±0.94aA 4.57±0.31cB 1.31±1.32cC Tricyclazole 88.28±0.34aA 81.43±0.17aA 70.87±0.38aA 5.10±1.04bB 1.63±1.03cC green health 88.12±0.26aA 81.30±0.15aA 69.87±0.58aA 7.30±1.21aA 2.63±0.54bB clear water 87.13±0.29bA 81.63±0.47aA 68.83±1.42aA 7.40±1.17aA 3.57±0.45aA

Note: The least range method (LSD) was used for significance analysis (P≤0.05±standard error).

(8) Antagonistic effect of Bacillus amyloliquefaciens S170 strain on various plant pathogenic bacteria:

Place Fusarium oxysporum Schl., F. solani and F. moniliforme fungus cakes in the center of the PDA medium plate. Bacillus S170 bacteria cake (5 mm) was inoculated at a distance of 2 cm from both sides of the edge of the colony, Rhizoctonia solani and strain S170 were cultured simultaneously, placed in an incubator at 28°C in the dark, and cultured 4 times. After 2 days, the diameter of the pathogenic bacteria colony and the zone of inhibition were measured, and the results are shown in Table 11.

Table 11 Antagonism of Bacillus amyloliquefaciens S170 strains to various plant pathogenic bacteria

pathogenic bacteria Antibacterial zone (mm) Mycelia growth inhibition rate (%) Fusarium oxysporum 6.5 48.15 Fusarium solani 3 33.89 Fusarium moniliforme 6 44.44 Rhizoctonia solani 9 67.23

(9) SEM observation of Bacillus amyloliquefaciens S170 strain inhibiting the infection structure of Magnaporthe grisea:

SEM observation of strain S170 on the mycelia inhibition of Magnaporthe grisea: activate Magnaporthe grisea on PDA solid medium, culture at 28°C for 5-7 days, and use a puncher with a diameter of 5mm to form bacteria on the activated Magnaporthe grisea plate cake. Use Magnaporthe grisea as the target bacterium, inoculate a cake of Magnaporthe grisea with a diameter of 5 mm in the center of the PDA plate, inoculate the bacteria to be tested at a distance of 2 cm from the target bacterial cake, and culture it in a 28°C incubator for 5 to 7 days. When the Mycelium of Magnaporthe grisea covered the entire Petri dish, take the Mycelium edge of the fungus Mycelia in the antibacterial zone on the confrontation culture plate and the Mycelium of the blank control (only grow fungus) at the corresponding position (cut together with the culture medium), and observe with a scanning electron microscope. Antibacterial zone, the results are shown in Figure 4.

SEM observation of the inhibition of conidia and hyphae by the sterile supernatant of bacterial strain S170: the sterile supernatant of the tested strain cultivated for 48 hours and the spore suspension of Magnaporthe grisea P131 (1×10 ⁶ CFU/mL) at a ratio of 1: Mix evenly at a ratio of 1, and incubate the mixture in a 2mL sterile centrifuge tube at 28°C for 24 hours. After high-speed refrigerated centrifugation, the precipitate is fixed with 2.5% glutaraldehyde for more than 2 hours, washed three times with phosphate buffer, and 1% starve acid. After fixing for 2 hours, rinse with phosphate buffer three times again, dehydrate three times with 30%, 50%, 70%, 80%, 90% and 100% ethanol gradient, dry with LEICA EM CPD critical point dryer, and spray gold with EIKO IB-3 , and finally adopted the Hitachi S-3400N scanning electron microscope to observe the impact of the tested strains on the conidia and mycelium morphology of Magnaporthe grisea, and the results are shown in Figure 5.

(10) Detection of biocontrol active substances of Bacillus amyloliquefaciens S170 strain

Detection of biofilm: Pick a single colony and inoculate it on liquid LB medium, shake it overnight at 30°C and 180rpm, add it to a 12-well tissue culture plate containing Msgg medium at a ratio of 1:1000, and culture it at 23°C. After 3 days, the formation of biofilm was observed, and the results are shown in Table 12.

Detection of amylase: inoculate a newly activated single colony on an LB plate containing 0.2% soluble starch, incubate for 48 hours, and after forming obvious colonies, stain the plate with Lugol’s iodine solution for 10 minutes, wash the plate with 70% ethanol , can produce amylase strains, on a black background, a colorless transparent circle can be formed around the place where the colony grows. If there is a transparent circle, it indicates that the strain can produce amylase. Each treatment was repeated 3 times, and the results are shown in Table 12.

Detection of protease: inoculate the activated strain to be tested on 1% skimmed milk agar plate, culture at 30°C for 24h, 48h, and 72h, and then observe the production of peripheral transparent circles. The appearance of transparent circles indicates the production of protease. Each treatment 3 replicated and the results are shown in Table 12.

Glucanase detection: The bacteria to be tested are inoculated on a plate containing ABP medium, and after culturing at 30°C for 48h and 72h, observe whether there is a digestion circle in the plate. If there is a digestion circle, it indicates the production of glucanase. Each treatment was repeated three times, and the results are shown in Table 12.

Siderophilic detection: Use CAS medium to detect siderophilicity. Inoculate the activated bacteria to be tested on the CAS detection medium plate, and after culturing at 30°C for 72 hours, observe whether there is an orange halo in the plate. Show siderophilic production, each treatment was repeated 3 times, the results are shown in Table 12.

Cellulase detection: activate the isolated strain and inoculate it on a cellulose screening medium plate, culture it upside down at a constant temperature of 28°C for 2 days, dip it with 0.1% Congo red staining solution for 10 minutes, and then decolorize it with 1mol/L NaCl solution 5min. If the bacterial strain produces cellulase, a clear transparent circle will appear around the colony. Each treatment was repeated 3 times. The results are shown in Table 12.

Detection of volatile substances: Inoculate a piece of Pyricularia grisea P131 bacterial block with a diameter of 1cm in the center of the tomato-oat medium plate, spread and cultivate the S170 strain on another NA medium plate of the same size, and place the former behind the latter Above, the contact between the two dishes was sealed with a parafilm, and the NA plate uncoated with the S170 strain was used as a blank control. After 9 days of constant temperature cultivation at 28°C, the colony diameters of the blast fungus coated with the S170 strain and the control culture were measured respectively, and calculated relative inhibition rate. 5 repetitions per treatment, the results are shown in Table 12 and Figure 7.

Preparation of Msgg medium: 0.1M Phosphate Buffer 50mL/L, 0.5M MOPS (pH 7.0) 200mL/L, 0.1M MgCl ₂ 20mL/L, 0.01M MnCl ₂ 5mL/L, 1mM ZnCl ₂ 1mL/L, 10mM Thiamine 0.2mL/L, 10mg/mL Phe 5mL/L, 10mg/mL Trp 5mL/L, 10mg/ml Thr 5mL/L, 50% glycerol 10mL/L, 10% Glutamate 50mL/L, deionized water 32mL/L, After filter sterilization, add sterilized 10mL 5mM FeCl ₃ and 7mL 0.1M CaCl ₂ , store at 4°C for use.

Production of ABP medium: use a mortar to grind lumpy Poria cocos into powder so that it can pass through a 120-mesh sieve, KH ₂ PO ₄ 6.8g, K ₂ PHO ₄ ·12H ₂ O 17.9g, yeast extract 6.7g, Poria powder (or β-1.3-glucan) 5.0g, aniline blue 120mg, agar powder 12g, H ₂ O 1L, pH value 6.8, water 1L.

Preparation of CAS medium: 9 g of caseytone, 5 g of yeast extract, 10 g of trisodium citrate, 1 g of disodium hydrogen phosphate, 1 g of sodium dihydrogen phosphate, 10 g of glucose, 20 g of agar, dissolved in 1 L of water.

Cellulose enriched medium (1L): NaCI 6g, MgS0 ₄ 0.1g, KH ₂ P0 ₄ 0.5g, CaCl ₂ 0.1g, (NH ₄ )S0 ₄ 2.0g, K ₂ HP0 ₄ 2.0g, agar 15g, CMC -Na 5g, pH adjusted to 7.0; Cellulose screening medium (1L): Add 1g of yeast powder to the above-mentioned cellulase enrichment medium.

Congo red staining solution: Dissolve Congo red in distilled water to a final concentration of 0.1% (w/v).

Congo red decolorization solution: NaCl solution with a final concentration of 1mol/L.

Table 12 Detection of biocontrol active substances of Bacillus amyloliquefaciens S170 strain

Note: The data are the detection results of three repetitions; "+" is the detection result, "-" is not detected

The applicant declares that the present invention illustrates the detailed features and methods of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed features and methods, that is, it does not mean that the present invention must rely on the above-mentioned detailed features and methods to be implemented. Those skilled in the art should understand that any improvement to the present invention, the equivalent replacement of the conditions selected in the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

Claims (7)

1. A Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) S170, preserved in the General Microorganism Center (CGMCC) of the China Microbiological Culture Collection Management Committee (CGMCC), the preservation date is May 22, 2017, and the preservation number is CGMCCNo.14179. 2. A bacterium agent, which comprises Bacillus amyloliquefaciens S170 as claimed in claim 1 or the sterile supernatant obtained by cultivating said Bacillus amyloliquefaciens S170. 3. A method for controlling rice blast, comprising applying Bacillus amyloliquefaciens S170 as claimed in claim 1 or the bacterial agent as claimed in claim 2 to rice crops. 4. The method according to claim 3, characterized in that, the bacterial agent is the culture solution of the Bacillus amyloliquefaciens S170. 5. The method according to claim 3 or 4, characterized in that the rice blast is leaf blast and/or neck blast. 6. the purposes of bacillus amyloliquefaciens S170 as claimed in claim 1 or bacterial agent as claimed in claim 2 in the prevention and treatment of rice blast. 7. The use according to claim 6, characterized in that the rice blast is leaf blast and/or neck blast.